Pharmacodynamic monitoring of canine T-cell cytokine responses to oral cyclosporine.

BACKGROUND: Pharmacodynamic assays measure the immunosuppressive effects of cyclosporine on T-cells and offer an alternative assessment of efficacy in individual patients. OBJECTIVE: To assess the immunosuppressive effects of high and low dosage cyclosporine on canine T-cells and to develop a novel testing system for individualized dose adjustment. ANIMALS: Seven healthy female Walker hounds. METHODS: Experimental study using a paired comparison design. Flow cytometry was used to measure T-cell expression of IL-2, IL-4, and IFN-γ. Cytokine expression 8 days after oral administration of high and low dosages of cyclosporine was compared to baseline and washout values, respectively. The high dosage was initially 10 mg/kg q12h and was then adjusted to attain established immunosuppressive trough blood drug concentrations (>600 ng/mL). The low dosage was 5 mg/kg q24h. RESULTS: High dosage cyclosporine resulted in significant decreases in IL-2 and IFN-γ expression (P = .0156, P = .0156), but not IL-4 expression (P = .2188). Low dosage cyclosporine was associated with a significant decrease in IFN-γ expression (P = .0156), while IL-2 expression was not affected (P = .1094). CONCLUSIONS AND CLINICAL IMPORTANCE: T-cell function is suppressed at trough blood drug concentrations exceeding 600 ng/mL, and is at least partially suppressed in some dogs at low dosages. Direct evaluation of T-cell function could be an effective, more sensitive alternative to measuring blood drug concentrations for monitoring immunosuppressive therapy.

Cyclosporine A affects the in vitro expression of T cell activation-related molecules and cytokines in dogs.

Cyclosporine is a powerful immunosuppressive drug that is being used with increasing frequency to treat a wide range of immune-mediated diseases in the dog. To date, ideal dosing protocols that will achieve immunosuppression with cyclosporine in dogs remain unclear, and standard methods that can measure effectiveness of immunosuppression have not been established. The aim of our study was to evaluate the effects of in vitro cyclosporine exposure on a panel of molecules expressed by activated T cells to ascertain their potential as biomarkers of immunosuppression in dogs. Blood was drawn from six healthy dogs, and peripheral blood mononuclear cells (PBMC) were isolated and activated. Half of the cells were incubated with 200 ng/mL cyclosporine prior to activation, and the other half were not exposed to cyclosporine. Samples were analyzed using flow cytometry, and the expression of intracellular cytokines IL-2, IL-4, and IFN-γ was evaluated after 6, 12, and 24h of drug exposure. Each cytokine exhibited a time-dependent suppression profile, and all but two samples activated in the presence of cyclosporine showed lower cytokine expression than untreated controls. We also evaluated the expression of the surface T cell activation molecules CD25 and CD95 by flow cytometry after 36 h of drug exposure. Expression of these surface molecules decreased significantly when activated in the presence of cyclosporine. Our results suggest that suppressed expression of the markers related to T cell activation could potentially be utilized as an indicator of the efficacy of cyclosporine therapy in dogs.

Bovine viral diarrhea viruses modulate toll-like receptors, cytokines and co-stimulatory molecules genes expression in bovine peripheral blood monocytes.

We have used noncytopathic (ncp) and cytopathic (cp) Bovine Viral Diarrhea Viruses (BVDV) to determine the expression levels of TLR genes, type I IFN, pro-inflammatory and Th1/Th2 cytokine gene expression in bovine monocytes. In general, both BVDV strains had similar effects. However, we found some significant differences that could be due to biological differences between cp and ncp BVDV strains. TLR3 was significantly up-regulated in 1h ncp, but not in cp BVDV- infected monocytes, whereas TLR7 expression dominated in 24h infection with both BVDV strains. Type I IFN and IL-12 gene expression was also significantly up-regulated in 1h ncp, but not cp BVDV infection that correlated with the enhanced TLR3 gene expression. Both BVDV biotypes suppressed pro-inflammatory cytokines TNF-alpha, IL-1beta, and IL-6, co-stimulatory molecules CD80 and CD86, but did not change Th1 type cytokine IL-12 and INF-gamma, gene expression after 24h infection. We hypothesize that BVDV may escape immune responses by altering the expression of TLR 3 and 7 and their signaling pathways.

Differential detergent fractionation for non-electrophoretic bovine peripheral blood monocyte proteomics reveals proteins involved in professional antigen presentation.

Professional antigen presenting cells (APC), dendritic cells (DC) and their myeloid progenitors, monocytes/macrophages are critical controllers of innate and adaptive immunity. Here we show that differential detergent fractionation (DDF) analysis of bovine monocytes reveals proteins related to antigen pattern recognition, uptake and presentation to immunocompetent lymphocytes. We identify 53 bovine proteins involved in immune function of professional APC. In particular, 13 adhesion molecules, three toll-like receptors (TLR1, 6 and 8), three antigen uptake-related proteins (including mannose receptor [MR] precursor), and eight actin-like proteins involved in active endocytosis were identified. In addition, MHC class I and II-related proteins, cytokines, active substances and growth factors have been identified. We conclude that the DDF approach can provide interpretable and meaningful functional information concerning protein expression profiles associated with monocyte activation, transformation into macrophages and/or immature DC, and maturation of monocyte-derived DC in the presence of multiple bovine pathogens.

Bovine monocytes induce immunoglobulin production in peripheral blood B lymphocytes.

Although a role for monocytes and monocyte-derived dendritic cells (DC) in the activation of T cells is well established, it is less clear to what extent DC and their precursors, monocytes, regulate B cell immune responses. Here we show that regulatory mechanisms similar to those in humans are in place in the bovine immune system. In vitro culture of bovine monocytes with bovine B cells activated by the anti-CD3 triggered CD4+ T cells or through immunoglobulin (Ig) receptor crosslinking induces B cell Ig secretion. Unlike bovine monocyte-derived DC, monocytes do not promote Ig class switching to IgG and IgA in activated peripheral blood B cells. These results suggest that bovine monocytes are capable of directly inducing Ig secretion in activated bovine peripheral blood B cells, but do not provide the signals for B cell Ig class switching.

Bovine dendritic cells generated from monocytes and bone marrow progenitors regulate immunoglobulin production in peripheral blood B cells.

We examined whether bovine monocyte-derived and bone marrow (BM) dendritic cells (DCs) regulate antibody production in activated peripheral blood B cells. DCs were generated from monocytes and BM progenitors in the presence of bovine recombinant granulocyte/macrophage colony-stimulating factor (GM-CSF) and interleukin 4 (IL-4). Monocyte-derived DCs promoted B cells activated by the anti-CD3 triggered CD4(+) T cells or through immunoglobulin M (IgM) receptor to increase the level of IgG secretion. Furthermore, the addition of DCs triggered B cells activated through IgM receptors to produce IgG2 and IgA, thus inducing an isotype switch. BM-derived DCs increased the production of IgG in B cells activated by the anti-CD3 triggered CD4(+) T cells, but unlike monocyte-derived DCs did not have any effect on B cells activated through surface IgM. These data suggest that the regulation of humoral immune responses in cattle depends on the origin of DCs and the mode of B cell activation.

Isolation and characterization of macaque dendritic cells from CD34(+) bone marrow progenitors.

We have developed a method for isolating and characterizing pigtailed macaque dendritic cells (DCs) generated from CD34(+) bone marrow (BM) progenitors based on methods previously developed for isolating human DCs. Macaque DCs displayed a characteristic morphology and were potent stimulators of allogeneic T cell proliferation. They expressed a set of DC-associated markers, such as MHC class II, CD1a, CD4, CD11a, CD40, CD58, CD80, CD83, CD86, and CXCR4. Macaque DCs, as well as peripheral blood CD4(+) T cells, were highly susceptible to HIV-2 infection, as detected by DNA-PCR. The expression of HIV-2 in macaque DCs was downregulated by treatment with the beta-chemokine RANTES. Macaque DCs will be useful for defining the in vivo role of DCs in HIV pathogenesis and for optimizing and testing peptide-DC vaccines or tolerizing regimens.

Nuclear import of HIV-1 DNA in resting CD4+ T cells requires a cyclosporin A-sensitive pathway.

Using PCR to monitor early (LTR/LTR (long terminal repeat)) and late (LTR/gag) products of reverse transcription and the formation of HIV-1 LTR circles (indicating nuclear import), we explored the relationship between T cell activation signals and early events in the life cycle of HIV-1 infection. The combination of TCR ligation with either CD28 cross-linking or exogenous IL-2 was required for HIV-1 LTR circle formation in resting CD4+ T cells. Ligation of the TCR or CD28 receptors or addition of IL-2 alone did not induce this process. However, cross-linking the TCR, or IL-2 alone, unlike CD28 ligation, could induce the completion of viral reverse transcription. In contrast, the initiation of HIV-1 reverse transcription could occur in resting CD4+ T cells without any stimulation. Cyclosporin A (CsA), an inhibitor of T cell activation, completely blocked HIV-1 DNA nuclear import in activated CD4+ T cells. The completion of HIV-1 reverse transcription was blocked by CsA in infected CD4+ T cells activated by TCR ligation and IL-2, but not in cells stimulated by TCR and CD28 ligation. The costimulation of CD3 and CD28 mAbs in the presence of IL-2 could not overcome the CsA inhibitory effect on nuclear import of viral DNA. Therefore, the factor(s) involved in a CsA-sensitive pathway plays a critical role in HIV-1 DNA transport from the cytoplasm into the nucleus. Production and movement of HIV-1 DNA in resting CD4+ T cells require two signals: one signal from the TCR, which normally regulates the G0 to G1 transition, induces completion of viral reverse transcription; the other signal through CD28 or an IL-2R-dependent process is sensitive to CsA treatment and regulates viral DNA entry into the nucleus.

Functional CD40 ligand expressed by human blood dendritic cells is up-regulated by CD40 ligation.

We examined whether human blood dendritic cells (DCs) express a functional ligand for CD40 (CD40L). Human blood DCs expressed significant amounts of cell surface CD40L identical to that expressed on activated T cells, as detected by mAb to CD40L or a chimeric CD40.Ig fusion protein (CD40.Ig). Stimulation through CD40 up-regulated protein and mRNA CD40L expression in DCs, B cells, and B cell lines. CD40-mediated CD40L expression was inhibited by a protein tyrosine kinase inhibitor, herbimycin, in a dose-dependent manner, suggesting that the induction of CD40L expression via CD40 requires protein tyrosine kinase activity. CD40L surface expression correlated with constitutive or inducible levels of CD40L-specific mRNA, as determined by reverse transcribed PCR analysis (RT-PCR) using CD40L-homologous primers. Furthermore, CD40L on DCs was functional, since CD40L+ DCs, unlike CD40L- DCs, induced B cell IgG and IgA production, and this induction could be inhibited by blocking CD40L-CD40 interactions with mAb to CD40L. Thus, CD40L on DCs and CD40L induced by crosslinking CD40 may regulate B cell activation and maturation. The importance of DC CD40L expression on B cell function is discussed.

Immunodeficiency virus cDNA synthesis in resting T lymphocytes is regulated by T cell activation signals and dendritic cells.

We explored the relationship between T cell activation signals and dendritic cells (DC) in the replication cycle of immunodeficiency viruses. First we analyzed the effect of two cell cycle inhibitors (mimosine and aphidicolin) on SIV reverse transcription, circularization, and integration in macaque resting T cells stimulated with anti-CD3 mAb at the time of infection. The formation of SIV LTR circles was blocked by the G1 inhibitor mimosine. The G1/S inhibitor aphidicolin neither affected circularization nor integration of SIV DNA. Therefore, the induction of SIV LTR circle production is likely to be mediated by signaling events normally regulating the G1 to S transition. We further characterized DC-dependent HIV-expression in human T cells. We examined the effect of ligating two novel receptors, IPO-3 and Bgp95, on DC-dependent HIV-1 expression. Activation of DCs through IPO-3 receptors, and to a lesser extent Bgp95 ligation, upregulated HIV spread in these cells. The mechanisms by which IPO-3 vs. Bgp95 increase HIV-1 levels appear to be different. In particular, IPO-3 ligation alone on T cells also increased HIV-1 levels. Activation of T cells via defined surface receptors or with DCs is required for establishing HIV/SIV cDNA synthesis in T cells.

Cell-cell interactions regulate dendritic cell-dependent HIV-1 production in CD4+ T lymphocytes.

We investigated the role of blood dendritic cells (DC) in transmission of HIV-1 from infected to uninfected CD4+ T cells, and the accessory molecules involved. DC promoted transmission from infected to uninfected CD4+ cells, but blood DC themselves were not infectable. DC-mediated transmission was blocked by mAb to CD4 and MHC class II, but strongly increased by mAb to CD40 on DC or CD28 on T cells. The DC-dependent infection was inhibitable by anti-CD80 and a soluble fusion protein of the CD80 ligand, CTLA4; soluble CTLA4Ig also blocked infection augmented by crosslinking CD40. We also demonstrated that mAb to CD40 up-regulate the expression of CTLA4 ligands CD80 and B70/B7-2 (CD86) on DC. These data suggest that the dialog between CD40-CD40 ligand (CD40L) and CD28-CD80 counter-receptors on DC and T cells may be linked to HIV infection in vivo.

The role of CD40 and CD80 accessory cell molecules in dendritic cell-dependent HIV-1 infection.

We investigated the role of blood dendritic cells (DCs) in transmission of HIV-1 from infected to uninfected CD4+ T cells, and the accessory molecules involved. DCs promoted transmission from infected to uninfected CD4+ cells, but DCs themselves were not infectable. DC-mediated transmission was blocked by MAb to CD4 and MHC class II, but strongly increased by MAb to CD40 on DCs or CD28 on T cells. The DC-dependent infection was inhibitable by anti-CD80 and a soluble fusion protein of the CD80 ligand, CTLA4; soluble CTLA4 immunoglobulin also blocked infection augmented by cross-linking CD40. These data suggest a linkage between CD40-CD40L and CD28-CD80 counterreceptors on DCs and T cells, and spread of HIV infection in vivo.

Costimulation through CD28 enhances T cell-dependent B cell activation via CD40-CD40L interaction.

Changes in T cell helper function were analyzed when anti-CD3-activated T cells were costimulated with mAbs to the CD28 receptor (anti-CD28). T cell-dependent B cell growth and differentiation were consistently augmented if anti-CD3 stimulated-T cells were simultaneously activated with anti-CD28. Although anti-CD28 enhanced IL-2 and IL-4 production, it did not increase B cell responses solely by augmenting production of soluble lymphokines. Anti-CD28 costimulation induced increases on T cells of CD40 ligand (CD40L), known to promote B cell proliferation and Ig secretion. Because anti-CD28 promoted T cell helper functions and expression of CD40L, we examined the dependence for CD40L during T cell-dependent B cell responses. Although soluble CD40 fusion proteins only partially inhibited T cell-dependent B cell activation, we found a strict requirement for CD40L expression at initiating B cell responses. Both CD40L expression and T cell help were blocked by cyclosporin A after TCR cross-linking, and, unlike T cell proliferation, both remained cyclosporin A sensitive during CD28 costimulation. In addition, anti-CD28 could not compensate for the T cell helper deficiency of hyper IgM syndrome patients who lack functional CD40L. Thus, anti-CD28-induced T cell help is delivered via a CD40L-dependent process. The fact that cross-linking CD40 on B cells promotes expression of the B7/BB-1 ligand for CD28 suggest T and B interactions may have a reciprocal amplification mechanism.

[Identification of cultures of Mycobacteria cultivated using sodium salicylate medium]. / Identifikatsiia kul'tur mikobakterii, vyrosshikh na srede s salitsilatom natriia.

Sodium salicylate test is used in bacteriological practice to distinguish M. tuberculosis and M. bovis from other mycobacterial species. The species of the strains growing in sodium salicylate media are not as a rule identified. This was done using gas chromatography with cultures which had grown in Lowenstein-Jensen media with sodium salicylate and isolated from the patients admitted to the Tuberculous hospital in 1991-1993. Only 20% of the strains belonged to human mycobacteria, the others were opportunistic or saprophyte mycobacteria (M. avium, M. fortuitum, M. phlei, M. flavescens, M. vaccae, M. smegmatis), some of the cultures were not of the Mycobacterium genus, but belonged to other Actinomycetales. It is evident that with sodium salicylate test one cannot be absolutely sure of Mycobacteria isolation. The strains growing in sodium salicylate medium call for further investigations.

[Phagocytosis and the antiphagocytic factors in mycobacterial infections]. / Fagotsitoz i antifagotsitarnye faktory pri mikobakterial'nykh infektsiiakh.

Peculiarities of phagocytosis of pathogenic, conditionally pathogenic ristic is given to antiphagocytic factors which permit some species of these processes with immunity are considered in the paper. A characteristic is given to antiphagocytic factors which permit some species of mycobacteria to avoid protective bactericidal reactions of mammals and to reproduce in phagocytes. Expression of mycobacterial virulence depends on the character of interrelations between the pathogen and cells of reticuloendothelial and immune systems. Interpretation of this mechanism is important in development of the methods of infection control and creation of efficient vaccines.

[Identification of Mycobacteria species by liquid-gas chromatography]. / Identifikatsiia vida mikobakterii metodom gazozhidkostnoi khromatografii.

Principles and characteristic features of gas chromatographic indentification of Mycobacterium from the fatty acid composition were considered. The results of their differentiation by gas chromatography agree with Myco. indentification according to the complex of morphological, cultural and biochemical, and other characteristics. Copyrolysis has a number of advantages compared to the routine identification techniques, i.e. it reduces the time needed for the analysis down to 2-3 hours; allows one to use small portions of microbial mass; and provides for a complete automation of the procedure. The advisability of putting the gas chromatographic identification of Myco. species into practice of the antituberculosis medical and veterinary institutions is pointed out.